Phosphoinositide signaling regulates all eukaryotic cells. In the phosphoinositide cycle, phosphatidylinositol (PI) is sequentially phosphorylated on the fourth and fifth hydroxyl of the myo-inositol ring by PI kinases and then phosphatidylinositol-phosphate kinases (PIPKs), forming phosphatidylinositol-4,5-bisphosphate (PIP2). PIP2 is a direct lipid messenger for many cellular responses and is an essential precursor to many other phosphatidylinositol-derived second messengers. Remarkably phosphoinositide signaling occurs within the nucleus where PIP2 is spatially generated at structures called nuclear speckles. Speckles have no identified membrane, but contain proteins and enzymes with roles in mRNA processing. PIPKs signal by interacting with effectors of the PIPn that they generate. PIPKI1 is an isoform that makes PIP2 that is present in the nucleus at speckles. PIP2 is also generated at speckles and may regulate activities of mRNA processing enzymes. In this context, we have discovered that PIPKI1 interacts with a novel poly A polymerase (PAP) now called Star-PAP. Star-PAP is dramatically and specifically stimulated by PIP2. Star-PAP and PIPKI1 are regulated by oxidative stress response and this regulates the expression of stress response mRNAs, including heme oxygenase-1 (HO-1), apolipoprotein E (APOE) and NAD(P)H:quinone oxidoreductase (NQO1). We hypothesize that PIPKI1 and Star-PAP function as a polyadenylation complex that associates with the transcriptional machinery required for 3'-processing of pre-mRNA transcripts. This novel polyadenylation complex is uniquely regulated by phosphoinositide signals and is required in vivo for 3'- processing of select mRNA transcripts, resulting in a novel mechanism to regulate gene expression. We will test this hypothesis with the following focused specific aims: 1. Study enzymatic activity of Star- PAP and regulation by phosphoinositides. Functional domains in Star-PAP that modulate specificity toward RNA substrates will be revealed. 2. Characterize Star-PAP complex assembly down stream of oxidative stress signals. The role of functional domains in Star-PAP will be defined. 3. The mechanisms for Star-PAP regulation of mRNAs in vivo will be investigated with an emphasis on the interaction with genes and mRNAs. It will be determined if the poly(A) tail generated by Star-PAP different than that by canonical PAP. The regulation of expression of the stress response proteins HO-1, APOE, and NQO1 play key biological roles that have dramatic implications for many aspects of human health including cardiovascular disease, transplantation, neurodegeneration and stroke, cancer therapy, and pulmonary medicine. The regulation of pre-mRNA polyadenylation by phosphoinositide signals via PIPKI1 and Star-PAP is an incredibly novel finding that has many implications for nuclear signal transduction and gene expression. Public Health Relevance: The expression of cellular proteins from genes occurs through messenger RNAs (mRNAs) made by each gene and this requires that the mRNA have a polyadenosine tail. This tail is required before the mRNA can make cellular proteins. We have discovered a new enzyme that uniquely makes these tails and this enzyme works to generate mRNAs and the encoded proteins. Most interesting and paradigm shifting is the fact that this process is regulated by a lipid messenger called phosphatidylinositol-4,5-bisphosphate. The genes whose expression this novel pathway controls are heme oxygenase-1 (HO-1), apolipoprotein E (APOE) and NAD(P)H:quinone oxidoreductase (NQO1). The control of these genes has dramatic implications for many aspects of human health including cardiovascular disease, transplantation, neurodegeneration, cancer therapy, and pulmonary medicine.